Mutant bacterial strains of the genus Sphingomonas deficient in production of polyhydroxybutyrate and a process of clarification of sphingans and compositions thereof

ABSTRACT

The invention relates to mutant strains of the genus  Sphingomonas  which have a mutation in at least one gene encoding a protein involved in polyhydroxybutyrate (“PHB”) synthesis that allows the mutant strains to produce PHB-deficient Sphingans. The invention is also directed to a process for preparing a clarified Sphingan solution comprising heating aqueous Sphingan solution, in particular PHB-deficient Sphingan solution, to a clarification temperature of about 30° C. to about 70° C., and treating the solution with a clarification agent and enzymes. In addition, the invention is directed to a food or industrial product comprising a PHB-deficient and/or clarified Sphingan. One particular embodiment of the invention is directed to a clarified, PHB-deficient high-acyl gellan and the processes of making thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/292,356 filed Dec. 2,2005, now U.S. Pat. No. 7,829,697 issued Nov. 9, 2010, which is adivisional of application Ser. No. 09/798,642 filed Mar. 2, 2001, nowU.S. Pat. No. 7,887,866 issued Feb. 15, 2011, which claims the benefitof U.S. Provisional Patent Application No. 60/186,433, filed Match 2,2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to mutant bacterial strains of the genusSphingomonas that are deficient in production of an internal storagepolymer, polyhydroxybutyrate (“PHB”) due to a null mutation, but producenormal quality of the capsular polysaccharides commonly referred to asSphingans. The present invention also relates to a method of clarifyingthe Sphingans produced by a mutant strain of Sphingomonas that isdeficient in the production of PHB. The present invention furtherrelates to food or industrial products comprising PHB-deficient and/orclarified Sphingans.

2. Discussion of the Related Art

Sphingans are capsular polysaccharides secreted by bacteria of the genusSphingomornas. Sphingans are structurally related, but not identical.Common members of the genus Sphingomonas and the Sphingans they produceinclude Sphingomonas elodea, ATCC 31461, which produces gellan (S-60);Sphingomonas sp. ATCC 31555, which produces welan (S-130); Sphingomonassp. ATCC 31961, which produces rhamsan (S-194); Sphingomonas s.p. ATCC53159, which produces diutan (S-657); Sphingomonas sp. ATCC 31554, whichproduces an as yet unnamed polysaccharide (S-88); Sphingomonas sp. ATCC31853, which produces an as yet unnamed polysaccharide (S-198);Sphingomonas sp. ATCC 21423, which produces an as yet unnamedpolysaccharide (S-7); Sphingomonas sp. ATCC 53272, which produces an asyet unnamed polysaccharide (NW-11); Sphingomonas sp. FERM-BP2015(previously Alcaligenes latus B-16), which produces alcalan (BiopolymerB-16) and the like. A description of the Sphingomonads and thepolysaccharides they produce can be found in U.S. Pat. Nos. 4,377,636;4,326,053; 4,326,052 and 4,385,123 (for ATCC 31461 and its S-60polysaccharide); in U.S. Pat. No. 4,342,866 (for ATCC 31555 and S-130);in U.S. Pat. No. 4,401,760 (for ATCC 31961 and S-194); in U.S. Pat. No.5,175,278 (for ATCC 53159 and S-657); in U.S. Pat. Nos. 4,331,440 and4,535,153 (for ATCC 31554 and S-88); in U.S. Pat. No. 4,529,797 (forATCC 31853 and S-198); in U.S. Pat. No. 3,960,832 (for ATCC 21423 andS-7); in U.S. Pat. No. 4,874,044 (for ATCC 53272 and NW-11); in U.S.Pat. No. 5,175,279 (for FERM BP-2015 and B-16), all of which areincorporated by reference herein.

Sphingan polysaccharides are structurally related by the primarystructure of their backbone, which comprises the sugars D-glucose,D-glucuronic acid, and L-rhamnose (or L-mannose). For example, theprimary structure of gellan, S-60, comprises the sugars D-glucose,D-glucuronic acid and L-rhamnose in a 2:1:1 molar ratio, which arelinked together to form a tetrasaccharide repeat unit in the followingorder: glucose, glucuronic acid, glucose, rhamnose. In the native form,gellan is modified by acetyl and glyceryl substituents on the sameglucose residue. On average, gellan has one glycerate substituent pertetrasaccharide repeat unit and one acetate substituent per every twotetrasaccharide repeat units. The primary structure of another Sphingan,diutan, S-657, differs from gellan in that it has an additionaldisaccharide side chain of L-rhamnose attached to one glucose residue,thus forming a hexapolysaccharide repeat unit. S-657 contains acetylgroups at position 2 and/or position 6 of the other glucose residue.

Sphingan polysaccharides, which are also referred to as gums, areprimarily used to thicken or gel aqueous solutions and are frequentlyclassified into two groups: thickeners and gelling agents. Typicalthickeners include starches, guar gum, carboxymethylcellulose, alginate,methylcellulose, xanthan, gum karaya and gum tragacanth. Common gellingagents include gellan, gelatin, starch, alginate, pectin, carrageenan,agar and methylcellulose.

Gelling agents are used in the food industry in a variety ofapplications, including confectionery jellies, jams, dessert gels,icings, dairy products, beverages and the like. Additionally, gellingagents may be used as components of microbiological media. Gellingagents differ in the conditions under which they may be used and in thetexture of the gels they form. These distinctive properties of gels haveled to the exclusive use of certain gelling agents in particularproducts (e.g., starch in confectionery jellies; gelatin in dessertgels; agar in icings; and alginate in pimento strips).

Despite the use of certain gelling agents in particular products,disadvantages exist for conventional food formulations. For example,gelatin, which is frequently used in dessert gel formulations, isanimal-sourced, requires refrigeration to set and is limited inapplication due to its instability under heat. Carrageenan, carrageenanand locust bean gum blends, and pectin, which are frequently used indessert gel, confectionery and jam/jelly formulations, are generallylimited to formulations that are brittle and inelastic in texture,suffer from poor storage stability and may be geographically restrictedfrom use in some countries, such as Japan. Starch, which is frequentlyused in confection formulations, provides poor clarity and poor flavorrelease. Consequently, it would be desirable to develop a gelling agentfor use in food formulations that is free from the problems associatedwith conventional gelling agents.

One particularly useful gelling agent is gellan (S-60), which is acapsular polysaccharide produced by the bacterium Sphingomonas elodea,ATCC 31461. Commercially, the gum is formed by inoculating afermentation medium under aerobic conditions with Sphingomonas elodeabacteria. The fermentation medium contains a carbon source, phosphate,organic and inorganic nitrogen sources and appropriate trace elements.The fermentation is conducted under sterile conditions with strictcontrol of aeration, agitation, temperature and pH. Upon completion ofthe fermentation, the viscous broth is pasteurized to kill viable cellsprior to recovery of the gum. However, the optimal fermentationconditions for producing gellan also promote production of the internalstorage polymer, polyhydroxybutyrate (“PHB”), which interferes with theultimate clarification and recovery of gellan. During fermentation, PHBsynthesis and gellan synthesis compete for the available carbon source,and PHB synthesis may compete with gellan synthesis.

Gellan displays different characteristics depending upon the method ofrecovery from the fermentation broth. Direct recovery from thefermentation broth yields gellan in its native or high-acyl form, whichis modified by S. elodea with acetyl and glyceryl substituents on oneglucose residue. Isolation of gellan in this native or high-acyl formyields a soft, flexible, elastic gel. Gellan may be deacylated bytreatment with hot alkali, thereby providing gellan in its low acylform. Isolation of gellan in this deacylated form yields a hard, firm,brittle gel, which has limited commercial applications. Blends of nativeand deacylated gellan produce gels of intermediate texture.

Certain applications require clear gellan. Currently, however, onlydeacylated gellan can be clarified. During the deacylation process,gellan is treated with alkali at high temperature, which removes theacyl substituents from the gellan and lyses the S. elodea cells. Solidsand cell debris are then removed by filtration yielding a clear,non-acylated gellan. To date it has not been possible to clarify gellanin its native or high-acyl form via filtration due to the high settemperature (the temperature at which a gum forms a gel upon cooling)required and the capsular nature of the organism, which does not allowfacile separation of gellan from the S. elodea cells. For applicationsrequiring native gellan, S. elodea cells may be lysed chemically orenzymatically; however, the remaining PHB will be present in the finalproduct and renders the resulting solutions turbid, rather than clear.

In addition to the use of gellan as a gelling agent, other Sphinganpolysaccharides have also found useful commercial application. The S-657polysaccharide imparts significant pseudoplasticity to polar solventssuch as water, such that S-657 can act as a rheological modifier that iscapable of particle suspension, friction reduction, emulsion and foamstabilization, filter cake disposition and filtration control.Consequently, S-657 has found industrial utility as a Rheologicalmodifier in a variety of cementitious systems, as disclosed in U.S. Pat.No. 6,110,271, which is incorporated herein by reference.

In addition to impairing clarity, the PHB found in Sphingans affects therheological properties of their gums. In particular, the PHB in S-657gum affects the ability of the polysaccharide to modify rheology inporous medial flow environments such as oil fields, wherein rheologyplays a significant role in well-bore drilling, completion and workoverfluids. In addition, PHB residue in S-657 may cause damage duringreservoir formation and may reduce the productivity of wells. Thepresence of PHB furthermore limits the applicability of S-657 gum inhousehold and personal care products, in which appearance is critical toconsumer acceptance.

Accordingly, attempts have been made to eliminate PHIB production inSphingans. One way to alleviate the problem of interfering PHBproduction in Sphingomonas species has been to chemically induce arandom mutation into a strain that inhibits production of PHB, such asdescribed in U.S. Pat. No. 5,300,429, which discloses LPG-2, a mutantstrain of Sphingomonas elodea that inhibits the production of PHB, butremains capable of producing gellan. Sphingomonas elodea was formerlyknown as Pseudomonas elodea and refers to the same organism. The LPG-2strain is on deposit with the American Type Culture Collection anddesignated ATCC 53967. While the LPG-2 strain produces gellan, itsquality is inconsistent, presumably due to the additional mutation(s)which occur with chemical mutagenesis.

Genetic engineering is a more selective mutagenesis approach forgenerating null mutant strains of Sphingomonas deficient for productionof PHB. Genetic engineering permits selective mutation or deletion of agene within the PHB synthesis pathway, which in turn permits completeinhibition of PHB production without affecting the quality of gumproduction.

Consequently, it would be highly desirable to develop mutant strains ofSphingomonas that are deficient in their ability to synthesize PHB,while maximizing Sphingan production and, concomitantly, mitigating therequisite effort to remove PHB from Sphingans.

SUMMARY OF THE INVENTION

The invention relates to mutant strains of the genus Sphingomonaswherein at least one gene encoding a protein involved inpolyhydroxybutyrate (“PHB”) synthesis is selectively mutated or deletedsuch that the mutant strains produce Sphingans but not PHB.

Another embodiment of the invention is directed to isolated DNAsequences isolated from the DNA of multiple Sphingomonas species, i.e.from ATCC 31461 and 53159, that encodes the protein PHB synthase.

Another embodiment of the invention is directed to a process ofpreparing a PHB-deficient, clarified Sphingan comprising the steps offermenting a mutant strain of the genus Sphingomonas and clarifying thePHB-deficient Sphingan from a fermentation broth.

Still another embodiment of the invention is directed to a process forpreparing a clarified Sphingan solution comprising heating a Sphinganfermentation broth to a clarification temperature of about 30° C. toabout 70° C., treating the Sphingan fermentation broth with aclarification agent and then treating the fermentation broth withenzymes. Yet another embodiment of the invention is directed to aprocess of preparing a clarified Sphingan solution comprising the stepsof heating a Sphingan fermentation broth to a clarification temperatureof about 30° C. to about 70° C., treating the fermentation broth with achelating agent, treating the fermentation broth with a lysozyme enzyme,treating the fermentation broth with a caustic or oxidizing agent, andtreating the fermentation broth with a protease enzyme.

Another embodiment of the invention is directed to mutant strains ofSphingomonas elodea that permit the preparation of a clarified,PHB-deficient, high-acyl (native) gellan with high gel strength.

Still another embodiment of the invention is directed to a food orindustrial product comprising a PHB-deficient and/or clarified Sphingan.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts PHB synthase protein sequences from Rhizobium meliloti(U17227) (SEQ. ID NO: 1), Alcaligenes eutrophus (J05003) (SEQ ID NO: 2),Acinetobacter sp. strain RA3849 (L37761) (SEQ ID NO: 3), Rhodobacterspaeroides (L17049) (SEQ ID NO: 4) and Methylobacterium extorguens(L07893) (SEQ ID NO: 5) aligned using the software DNA star MegAlign® byLaserGene (Madison, Wis.). Regions I and II were selected as conservedregions with moderate degeneracy and positioned to provide a polymerasechain reaction (“PCR”) product of about 400 base pairs (“bp”).

FIG. 2 shows the sequence of the 408 bp insert in plasmid pEB1 (SEQ IDNO: 6).

FIG. 3 is a schematic illustrating the steps used to clone and constructan internal deletion in the Sphingomonas elodea phaC gene.

FIG. 4 depicts the sequence of the phaC region (SEQ ID NO: 7).Restriction enzyme sites for PstI (CTGCAG) are underlined. Primerbinding sites are indicated by arrows. A portion of phaC gene extendsfrom the first PstI site to the TGA stop codon (in bold). The bases thatare deleted in the mutants are set out separately. The XbaI site(TCTAGA, double underlined) is substituted for the deleted region in themutants, as described in the text.

FIG. 5 is a schematic diagram of homologous recombination of mutatedphaC gene into the Sphingomonas elodea chromosome and excision of theintegrated vector leaving either an intact or mutated phaC gene in thechromosome.

FIG. 6 is an illustration of the plasmid pLO2.

FIG. 7 is a schematic diagram demonstrating integration of a vectorcontaining a phaC deletion into a Sphingomonas elodea chromosome.

FIG. 8 is a graphical representation of cell counts determined byplating broth samples from 10 L fermentations.

FIG. 9 shows a Southern hybridization of Sphingomonas genomic DNApreparations digested with EcoRI and hybridized to a probe for the ATCC53159 phaC gene. Lanes 1 and 2 contain size markers (.lamda. HindIII and.lamda. HindIII+EcoRI, respectively). Lanes 3-6 contain genomic DNAdigests from Sphingomonas sp. strains ATCC 53159, 31461, 31555 and31961, respectively.

FIG. 10 is the DNA sequence of the phaC gene and flanking regions ofATCC 53159 (SEQ ID NO: 13). Restriction enzyme sites for BamHI (ggatc),EcoRI (gaattc) and NotI (gcggccgc) are underlined and the overlap primersites are double-underlined. Primer sites are indicated by arrows. ThephaC gene is highlighted in bold.

FIG. 11 depicts a genetic map of the phaC region and primers for PCRamplification.

FIG. 12 depicts the cloning strategy in which PCR was used to constructa product containing only the regions flanking phaC and omitting theentire phaC gene.

FIG. 13 is a graphical representation of the effect of potassiumhydroxide concentration on transmittance.

FIG. 14 is a graphical representation of the effect of potassiumhydroxide concentration on gel strength.

FIG. 15 is a graphical representation of the effect of Calgonconcentration on transmittance.

FIG. 16 is a graphical representation of the effect of Calgonconcentration on gel strength.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetically engineered strains of thegenus Sphingomonas deficient in their ability to synthesize the internalstorage polymer polyhydroxybutyrate (“PHB”) due to a null mutation whichinactivates PHB synthesis. The PHB-deficient mutant Sphingomonas strainsof this invention are capable of synthesizing commercially usefulSphingans which are free of PHB, as determined qualitatively byturbidimetric methods well known in the art (see example 4 below, andU.S. Pat. No. 5,300,429, the contents of which are incorporated byreference). PHB is a storage polymer that accumulates intracellularly inSphingomonas under conditions of high carbon and low nitrogen, which arethe same conditions that produce optimal levels of Sphingans.

PHB synthesis has been studied in a number of organisms, and at leastthree genes for PHB synthesis have been identified (Anderson, A. J. andE. A. Dawes, Microbiol. Rev 54: 450-72 (1990)). PHB is derived fromacetyl coenzyme A (CoA) in three steps. The first step is catalyzed by3-ketothiolase (phaA) and results in the formation of acetoacetyl CoA.In the second step, the enzyme acetoacetyl CoA reductase (phaB) convertsacetoacetyl CoA to .beta.-hydroxybutyryl CoA, which is finallypolymerized by PHB synthase (phaC) in the third step to form PHB. Amutation wherein at least one gene encoding a protein involved inpolyhydroxybutyrate synthesis, i.e. phaA, phaB, or phaC, is selectivelymutated or deleted may result in a PHB-deficient Sphingomonas strain.

For example, the Sphingomonas mutant strains described herein are theresult of at least two mutations: (1) a deletion of or within the phaCgene encoding PHB synthase to block production of PHB, which had theunexpected result of diminishing Sphingan production; and (2) aspontaneous mutation to restore Sphingan production. The presentinvention also provides an optional preliminary mutation comprising aspontaneous mutation to increase the ability of Sphingomonas mutants totake up plasmid DNA, i.e. the S-60 wtc mutation in Sphingomonas elodea.

Additionally, the present invention discloses a method of clarifyingPHB-deficient gellan and other Sphingans produced by mutant Sphingomonasstrains using chelating agents, caustic or oxidizing agents and enzymesfor cell lysis and protein digestion. The present invention alsodiscloses food or industrial products comprising PHB-deficient and/orclarified Sphingans.

To illustrate the details of the invention, the steps involved in thegenetic engineering of Sphingomonas elodea and Sphingomonas sp. ATCC53159 are described, however, as noted below, the invention is notlimited to engineering Sphingomonas elodea and Sphingomonas sp. ATCC53159 nor any particular gene encoding a protein involved in thesynthesis of PHB.

An internal fragment of the S. elodea strain, ATCC 31461, phaC gene wasobtained by PCR with degenerate primers designed from two conservedregions of phaC encoded proteins. The nucleotide sequence of thisfragment, as shown in FIG. 2, was utilized to design primers for inversePCR that allowed isolation of a larger portion of the phaC gene and 3′flanking sequence. Generally, the technique of inverse PCR clones theflanking regions of the nucleotides of interest in an orientationinverted to its natural orientation (See FIG. 3). The cloning processthat arranged the inverted PCR fragments in their natural orientationresulted in a deletion of 232 base pairs (“bp”). Allelic exchange ofthis fragment for the chromosomal phaC gene eliminated PHB production inS. elodea. The internal 232 bp deletion had the unexpected effect ofreducing gellan production. Spontaneous derivatives with restored gellanproduction were isolated from large scale growth of the mutant S.elodea. The PHR-deficient derivatives of the present invention containno foreign DNA, a deletion of 232 bp from the native chromosome and anuncharacterized spontaneous mutation. The PDG-1 and PDG-3 strains are ondeposit with the American Type Culture Collection and designated as ATCCNo. PTA-4863 and ATCC No. PTA-4864, respectively both deposited on Dec.20, 2002.

The particular molecular biology techniques, i.e. inverse PCR anddeletion mutations, used to generate the Sphingomonas mutant for PHBproduction are not critical. It is within the knowledge of one ofordinary skill in the art to use conventional molecular biologytechniques to generate Sphingomonas mutants. Other useful molecularbiology techniques that may be used to mutate phaC-like genes indifferent Sphingomonas strains include, but are not limited totransposon mutagenesis, point mutations and insertion element mutations.

The phaC gene is only one gene in the PHB synthesis pathway; thus it ispossible to generate Sphingomonas mutants with the desired phenotype,i.e., deficient in production of PHB, by selectively mutating ordeleting other genes involved in the PHB synthesis pathway. Genes ofinterest that may be selectively mutated to yield the desired phenotypeinclude, but are not limited to phaA (3-ketothiolase) and phaB(acetoacetyl CoA reductase).

Once the Sphingomonas mutants are generated, they are grown or fermentedin an aqueous solution known as a fermentation broth into which theSphingans are secreted as capsular polysaccharides. Followingfermentation of the PHB-deficient Sphingomonas mutants, the Sphingansmay be prepared by pasteurizing the broth and precipitating the Sphinganwith an alcohol such as isopropanol, using techniques well-known in theart.

Preferably, following fermentation, the Sphingans can be clarified andisolated away from the suspended solids and cellular debris that arepart of the fermentation broth milieu to yield PHB-deficient, clarifiedSphingans. In addition, the clarification process of this invention maybe applied to any Sphingan strain in addition to the above PHB-deficientSphingans. As described herein, the clarification process comprisesheating the fermentation broth and treating the fermentation broth withone or more chelating agents, one or more caustic or oxidizing agents,or a mixture thereof, followed by treatment with any lysozyme enzymesand/or any protease enzymes.

Specifically for gellan, the S. elodea mutant deficient in PHBproduction combined with the clarification process of this inventionenables the production of clarified gellan in its high-acyl form. Thegellan resulting from this mutant and process displays good clarity andhigh gel strength, which is useful for making dessert gels,confectionery, beverages and the like.

In one embodiment of this invention, hereinafter referred to as the“first protocol”, aqueous solutions of Sphingans may be clarified by aprocess comprising treating the Sphingan solution with one or moreoptional surfactants, one or more chelating agents, one or more causticor oxidizing agents, or a mixture thereof, and then treating with anylysozyme enzyme(s) and/or any protease enzyme(s).

In another embodiment of this invention, hereinafter referred to as the“second protocol” aqueous solutions of Sphingans may be clarified by aprocess comprising treating the Sphingan solution with one or morechelating agents, followed by any lysozyme enzyme(s), followed by one ormore caustic or oxidizing agent(s), followed by any protease enzyme(s)or a mixture of protease enzymes.

In the first protocol, the process of this invention may be conducted ina stepwise manner, wherein the Sphingan solution is first treated withthe chelating agent(s), optional surfactant(s), caustic or oxidizingagent(s) or a mixture thereof, and is then treated with any lysozymeenzyme(s) and/or any protease enzyme(s). In the second protocol, thestepwise process may be conducted wherein the Sphingan solution is firsttreated with the chelating agent(s), then any lysozyme enzyme(s), thenthe caustic or oxidizing agent(s) and then any protease enzyme(s), inthat order.

Advantageously, the process for producing clarified Sphingan solutionsdescribed herein provides Sphingan solutions that may be used, ifdesired, after appropriate dilution, without any further chemical ormechanical treatment (except for pasteurization and precipitation). Forsome applications, Sphingans may be isolated from these clarifiedSphingan broths by pasteurizing the broth, adjusting the broth to thedesired pH and precipitating the Sphingan with an alcohol (i.e.,isopropyl alcohol) according to conventional techniques.

Rehydration and dissolution of this Sphingan in water provides asubstantially clear Sphingan solution. A substantially clear Sphingansolution (1% w/w), according to this invention, has a lighttransmittance greater than about 60%, preferably greater than 70%, andmost preferably, greater than 80%. Light transmittance may be measuredat any wavelength in the visible spectrum using conventional techniquesand equipment (e.g., commercially available spectrophotometers). Thelight transmittance is typically measured at wavelengths of about 600 nmto about 650 nm. Light transmittance may be determined for several typesof Sphingan solutions: untreated broth, partially treated broth (e.g.,broth treated only with a chelating agent(s), a caustic or oxidizingagent(s), a chelating/caustic or chelating/oxidizing agent mixture, or abroth treated only with a lysozyme and/or protease enzyme), treatedbroth, or reconstituted Sphingan solutions. The substantially clearsolutions described herein, having a light transmittance greater thanabout 60%, are aqueous solutions containing about 1% by weight of theSphingan, isolated from a broth treated by the method according to thisinvention.

The Sphingan solutions that may be clarified using the process of thisinvention include the whole fermentation broth containing Sphingansobtained by fermentation of a Sphingan-producing microorganism in anutrient medium, solutions obtained by addition of isolated Sphingans toaqueous media and partially purified Sphingan solutions. The aqueoussolutions of Sphingans containing undesirable fermentation solids usefulin the process of this invention may contain about 0.01% to about 10%Sphingan by weight of the total weight of the solution. Any aqueoussolution containing any of the known Sphingans may be used in thepractice of this invention.

The first step of either clarification process of this inventioncomprises heating a Sphingan solution to a clarification temperature byconventional techniques, such as temperature control in a jacketed tank,direct steam injection, or the like. Direct steam injection is preferredto minimize heating time. The clarification temperature ranges fromabout 30° C. to about 70° C. and, preferably, from about 50° C. to about60° C. The length of time required to heat the Sphingan solution to thedesired temperature may vary significantly depending upon the size andvolume of the Sphingan solution to be treated. For example, whereas itmay take only several minutes to increase the temperature of a smallvolume (e.g., 50 ml) of Sphingan solution from room temperature to about60° C., it may take several hours to similarly increase the temperatureof 40,000 liters of solution (e.g., as may be present in batchprocessing).

The next step of the process of this invention comprises treating anaqueous Sphingan solution with a clarification agent selected from atleast one chelating agent, at least one caustic or oxidizing agent, or amixture thereof, according to one of the two protocols. Alternatively,the addition of a clarification agent may be conducted simultaneouslywith heating the Sphingan broth to the clarification temperaturedescribed above.

In the first protocol, the next step is the addition of the chelatingagent(s) to the Sphingan solution in the presence of caustic oroxidizing agent(s). Typically, the contact time for the chelatingagent(s) and caustic/oxidizing agent(s) ranges from about 0.5 hours toabout 2 hours each and, preferably, about 1 hour for the chelatingagent(s) and from about 0.5 hours to about 1.0 hours for the caustic oroxidizing agent(s). Typically, the caustic or oxidizing agent(s) isadded to the Sphingan solution at a concentration ranging from about 0g/L to about 2 g/L and, preferably from about 0.5 g/L to about 1.5 g/L.Typically, the chelating agent(s) is added to the Sphingan solution at aconcentration ranging from about 0 parts per million (“ppm”), to about3000 ppm and, preferably, from about 1000 ppm to about 2000 ppm.

After treatment with the clarification agent in this first protocol, theSphingan broth is subjected to an enzymatic treatment step, wherein theenzymes lysozyme and/or protease are added to the Sphingan broth eitherseparately or simultaneously. Typically, the enzymes are contacted withthe Sphingan broth for a time period ranging from at least about 0.5 hrto 8 hrs each, preferably at least 1 hr each, and most preferably atleast 2 hrs each. The typical lysozyme concentration ranges from about11,000 MCG units/L to about 44,000 MCG units/L, preferably, from about20,000 MCG units/L to about 25,000 MCG units/L; the typical proteaseconcentration ranges from about 65,000 Delft units/L to about 260,000Delft units/L, preferably, from about 100,000 Delft units/L to about150,000 Delft units/L. As used in this application, an “MCG unit” refersto a rate of lysis of Micrococcus lysodeikticus compared to a referencestandard at pH 6.6 and 37° C. as described by Genencor InternationalInc.; similarly, the term “Delft unit” refers to a specific assayinvolving the rate of extinction of a case in solution provided by thevendor Genencor.

The enzymes used in the enzymatic treatment step degrade the solidcellular debris to soluble compounds, thus improving transmittance ofthe Sphingan solution and aiding in the clarification process. Theprotease enzymes suitable for use in this process may be acid, neutralor alkaline proteases from bacterial, fungal or plant sources. Exemplaryacid protease enzymes useful in the process of this invention include,but are not limited to proteases produced by microorganisms of the genusAspergillus, such as A. niger. The neutral protease enzymes useful inthe process of this invention include, but are not limited to proteasessuch as Bacillus amyloliquifaciens. The alkaline protease enzymes usefulin the process of this invention include, but are not limited tomicroorganisms of the genus Bacillus, such as B. subtilis, B.licheniformis, and B. pumilis, proteases elaborated by species ofStreptomyces, such as S. fradiae, S. griseus and S. rectus, andproteases obtained from subtilisins, such as subtilisin Novo, subtilisinCarlsberg, including proteases such as subtilopeptidase A andsubtilopeptidase B. The lysozymes suitable for use in this processinclude the Multifect® lysozyme from Genencor International Inc.(Rochester, N.Y.) or any lysozyme that may be obtained from a plant,animal or microbially-derived source. The source of any of the proteaseenzymes or lysozymes used in the present invention is not critical.These enzymes and the methods of obtaining them are well known in theart.

As described above in the first protocol, the enzymes comprising theenzyme treatment (treatment with lysozyme enzymes and/or proteaseenzymes) may be added simultaneously or separately. Simultaneoustreatment refers to addition of the protease enzyme and lysozyme enzymeto the Sphingan solution in any order, over any period of time, providedthat both enzymes are present in the Sphingan solution during thetreatment. When added simultaneously, the enzyme treatment process ofthis invention is conducted under conditions such that both lysozymeenzymes and protease enzymes are active and provide the desiredenzymatic function. The simultaneous enzyme treatment process of thisembodiment may be conducted at a temperature of about 30° C. to about70° C. at a pH of about 5 to about 9, and preferably about 6 to about 8.While the specific temperature and pH range of this embodiment may varydepending on the enzymes used, in this simultaneous embodiment, theprocess of the present invention is conducted at relatively mildtemperatures and at nearly neutral conditions such that both thelysozyme enzyme and protease enzymes (acid, neutral or alkalineproteases) will demonstrate acceptable levels of activity to clarify theSphingan solution.

Preferably, the enzyme treatment is conducted such that any lysozymeand/or protease enzymes are each separately added to the Sphingansolution. Most preferably, each enzyme is separately added to theSphingan solution under its respective, optimal pH conditions, i.e. anacidic to neutral pH range for lysozyme (pH range of about 3 to about7.5), and a neutral to basic pH range for protease (pH range of about6.5 to about 9). The temperature and pH range at which differentlysozyme and protease enzymes demonstrate optimal clarification activitymay vary. Furthermore, if a choice must be made between lysozyme enzymesor protease enzymes for use in the enzyme treatment, then preferably theenzyme treatment comprises one or more protease enzyme(s).

In the second protocol, the chelating step is followed by enzymatictreatment with any lysozyme enzyme(s), which is followed by treatmentwith one or more caustic or oxidizing agent(s), followed by enzymatictreatment with any protease enzyme(s). As illustrated above, theenzymatic treatment is bifurcated between lysozyme enzyme(s) andprotease enzyme(s). This alternative sequence allows any lysozymeenzyme(s) to act under its preferred neutral to acidic pH conditions,and allows any protease enzyme(s) to act under its preferred neutral tobasic pH conditions. The same lysozyme and protease enzymes, andchelating, surfactant and caustic or oxidizing agents, may be used inpracticing the second protocol as described above in the first protocol.

Agitation of the Sphingan solution is not essential, although wherefeasible the Sphingan solution is stirred or agitated mildly orperiodically to avoid undue settling of the solids and promote contactwith the enzymes.

Chelating agents that are suitable for use in the process of thisinvention are compounds or compositions that are capable of sequesteringmultivalent metal ions (e.g., Mg⁺², Ca⁺², etc.) in the Sphingan solutionby forming poly-dentate complexes with the metal ions, forming aprecipitate with the metal ions or adsorbing the metal ions. Preferably,the chelating agents are water or water-alcohol soluble compounds orcompositions and are alkali metal or alkaline earth salts of organicand/or inorganic acids or organic/inorganic acid salts of basic(amine-containing) organic compounds, as well as the organic and/orinorganic acids or the basic compounds themselves. Other chelatingagents useful in the process of this invention are cationic ion exchangeresins and carbonic acid and carbonic acid salts. Salt compounds andcompositions that are particularly useful in the process of thisinvention include the salts of ethylenediamine tetraacetic acid,phosphoric acid, metaphosphoric acid, carbonic acid, citric acid,tartaric acid, gluconic acid, glutamic acid, pyrophosphoric acid,polyphosphoric acid, metaphosphoric acids, saccharic acid,ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid(EGTA), ethylenediamine, 2,3-diaminobutane, 1,2-diaminocyclohexane,triaminotriethylamine and the like. Useful salts may include the mono-,di-, tri- and/or tetra-metal salts of the above acids and the mono-, di-or tri-acid salts of the above bases, as appropriate. Preferably, thechelating agents used in the process of this invention include salts ofethylenediamine tetraacetic acid, citric acid, phosphoric acid,pyrophosphoric acid, polyphosphoric acid, carbonic acid, metaphosphoricacid, and ethylenediamine. Examples of useful chelating agents include,but are not limited to, disodium ethylenediamine tetraacetate,dipotassium ethylenediamine tetraacetate, tetrasodium ethylenediaminetetraacetate, tetrapotassium ethylenediamine tetraacetate, trisodiumcitrate, tripotassium citrate, sodium hexametaphosphate, potassiumhexametaphosphate, sodium polyphosphate, potassium polyphosphate, sodiumpyrophosphate, potassium pyrophosphate, monosodium phosphate,monopotassium phosphate, disodium phosphate, dipotassium phosphate,trisodium phosphate, tripotassium phosphate, sodium bicarbonate, sodiumcarbonate, potassium carbonate, potassium bicarbonate, a cationic ionexchange resin, ethylenediamine dihydrochloride, ethylenediaminediacetate, ethylenediamine lithium salt, ethylenediamine dihydroiodideand the like. More preferably, sodium hexametaphosphate is used as thechelating agent.

As described in the above protocols, surfactants may optionally be usedin conjunction with the caustic, oxidizing and chelating agents in orderto further improve transmittance in the final gellan product.Surfactants that are suitable for use in the process of this inventionare compounds or compositions that are capable of forming aqueousemulsions in the presence of hydrophilic and hydrophobic substances(solids or liquids). Preferably, the surfactants are water orwater-alcohol soluble compounds or compositions. Examples of usefulsurfactants include, but are not limited to SDS, polyoxyethylenesorbitanmonooleate (Tween 80® by ICI Americas, Inc., Bridgewater, N.J.) but arenot limited to SDS, lecithin, monoglycerides, tartaric esters ofmonoglycerides, phosphated monoglycerides (e.g., as the monosodiumsalt), lactylated monoglycerides, acetylated monoglycerides,succinylated monoglycerides, ethoxylated monoglycerides, sorbitanesters, polysorbates, polyglycerol esters, sucrose esters, sodiumstearoyl lactylate, propylene glycol esters and the like.

The optional surfactants are added to the Sphingan broth at any timeduring treatment with the chelating agent(s), caustic or oxidizingagent(s), for a contact time ranging from about 0.5 hours to about 8hours each and, preferably, about 2 hours. Typically, the surfactantsare added to the Sphingan solution at a concentration ranging from about0.0 g/L to about 3.0 g/L and, preferably from about 0.1 g/L to about 1.0g/L. Typically, the surfactant(s) is added to the Sphingan solution at aconcentration ranging from about 0 parts per million (“ppm”), to about3000 ppm and, preferably, from about 300 ppm to about 1000 ppm.

Caustic agents that are suitable for use in the process of thisinvention include, but are not limited to, potassium hydroxide, sodiumhydroxide, trisodium phosphate and the like. Potassium hydroxide is thepreferred caustic agent. Alternatively, oxidizing agents may be used inlieu of caustic agents. Oxidizing agents that may be used in theclarification process of the present invention include sodiumhypochlorite or other hypochlorite salts, chloride dioxide, hydrogenperoxide, peracetic acid, ozone, and other oxidizing agents well knownin the art. In the present invention, the preferred oxidizing agent issodium hypochlorite.

It should be noted that the degree of clarification effected bytreatment of the Sphingan solution with chelating agent(s),surfactant(s), caustic or oxidizing agent(s) or mixture thereof mayaffect the enzyme concentrations or the time required to complete thesubsequent enzyme treatment. For example, increasing the amount of thechelating agent(s), surfactant(s), caustic or oxidizing agent(s) or amixture thereof used in this process may decrease the amount of enzymesused and/or the time required to effect clarification of a Sphingansolution. Adjustment and balancing of the concentration and length oftreatment time of the chelating agent(s), surfactant(s), caustic oroxidizing agent(s) or mixture thereof and/or with the concentration andlength of treatment time of the lysozyme and/or protease to obtainSphingan solutions is preferable for optimizing production of thePHB-deficient, clarified Sphingans described herein.

The PHB-deficient and/or clarified Sphingans described herein may beused in a variety of food or industrial applications. For example, aPHB-deficient and/or clarified Sphingan such as native (high-acyl)gellan may be used to improve the taste, texture, stability andappearance of food products such as dessert gels, confections, jams andjellies, beverages, films, coatings and the like. As an additionalexample, a PHB-deficient and/or clarified Sphingan such as S-657 mayfind improved effectiveness as a rheological modifier in industrialapplications such as oil-field drilling or cementitious systems. OtherPHB-deficient and/or clarified Sphingans of the present invention willalso find a greater range of application in both food products andindustry.

The following examples provide illustrations of the present inventionand should not be misconstrued to limit in any way the scope of thepresent invention.

Example 1 Generation of a Sphingomonas elodea phaC Fragment

To identify highly conserved regions of the PHB synthase gene. PHBsynthase sequences from diverse organisms were retrieved from theNational Center for Biotechnology Information (“NCBI”) Gene Bank.Sequences from Rhizobium meliloti (gb: U17227) (SEQ ID NO: 1),Rhodobacter spaeroides (gb: L17049) (SEQ ID NO: 4), Methylobacteriumextorquens (gb: 07893) (SEQ ID NO: 5), Alcaligenes eutrophus (gb:J05003) (SEQ ID NO: 2) and Acinetobacter sp. strain RA3849 (gb: L37761)(SEQ ID NO: 3) were selected and studied. The protein sequences of theselected PHB synthase genes were aligned as displayed in FIG. 1. Amongthe conserved regions, Region I (R. meliloti codons 411-417) and RegionII (R. meliloti codons 535-541) were selected to provide a PCR productof about 408 bp based on their position and relatively low degree ofdegeneracy.

Degenerate PCR primers were designed to amplify the sequence betweenRegion I and Region II based on the conserved protein sequences and theapparent codon preference of Sphingomonas elodea, ATCC 31461. The codonpreference was inferred from the codon usage in five genes from theregion encoding exopolysaccharide biosynthetic enzymes in S. elodea,ATCC 31461, sequenced by Dr. Luis lelpi (unpublished) and from thecomplete exopolysaccharide biosynthetic enzyme gene cluster from theclosely related Sphingomonas ATCC 31554, which produces S-88 gum(Yamazaki, et al., J. Bacteriol. 178: 2676-2687 (1996)).

The N-terminal primer, designated PHADG5 (SEQ ID NO: 9), comprised a5′-AGTT clamp region, a TCTAGA XbaI site, and a TTC GAY CTS CTS TAY TGGAAY3′ degenerate hybridizing region targeting Region I. The C-terminalprimer, designated PHADG7 (SEQ ID NO: 10), comprised a 5′-GTAT clamp, aACTAGT SpeI site, and a CCA III SGG CCA CCA GCT GCC degenerate regiontargeting Region II. In SEQ ID NO: 10, “I” refers to inosine, anucleotide that is compatible with any other base, that is, A, C, T orG.

Primers PHADG5 (SEQ ID NO: 9) and PHADG7 (SEQ ID NO: 10) were utilizedin a PCR reaction with chromosomal DNA from a non-mucoid strain, Gps31,serving as the template. Gps31 is a non-gellan producing mutant of S-60.Taq polymerase with the Taq Start® Antibody by Clontech Laboratories,Inc. (Palo Alto, Calif.) provided a hot start for PCR with 2.5 mM eachdNTP, 4 mM MgCl.sub.2 and 50 pmol each primer in a reaction volume of100 .mu.l. The temperature program was 5 minutes 96° C., 30 cycles of 1min 96° C., 1 min 58° C., 1 min 72° C., and 5 min 72° C. before stoppingthe reaction by chilling to 4° C. The PCR reaction resulted in a singleband at the expected 416 bp size (408 bp plus clamps). Followingdigestion with XbaI and SpeI, the fragment was cloned into an XbaIdigested, calf intestinal alkaline phosphatase (“CIAP”) treated pUC19vector to yield plasmid pEB1. The DNA sequence of the 408 bp insert (SEQID NO: 6) from both strands is illustrated in FIG. 2. The fragmentcontained restriction sites for EcoRI, KpnI and PvuII. An alignment ofthe translated cloned fragment with other PHB synthase proteinsdemonstrated that a PHB synthase had been cloned.

Example 2 Construction of phaC Deletion by Inverse PCR

Southern hybridization was used to determine an appropriate restrictionenzyme that would provide a larger fragment of the Sphingomonas S-60phaC gene that was still not too large for facile recovery by inversePCR. Chromosomal DNA was isolated from Gps31 according to the methoddescribed in the QIAGEN® (Valencia, Calif.) DNA purification kit. ASouthern analysis using a probe generated from the 408 bp insert (SEQ IDNO: 6) cloned in pEB1 demonstrated that in a PstI digest of S. elodeaDNA, the 408 bp phaC fragment (SEQ ID NO: 6) resided on a fragment ofabout 2 kb.

The sequence of the 408 bp Sphingomonas S-60 phaC fragment (SEQ ID NO:6) was used to select outward reading PCR primers, as illustrated inFIG. 2. Primer PHAC12 (SEQ ID NO: 11) reads toward the N-terminal end ofthe phaC encoded protein with a clamp 5′GTTC, an XbaI site TCTAGA, andhybridizing region GGC GCG ATC AGC TTG TTG TC3′. Primer PHAC11 (SEQ IDNO: 12) reads toward the C-terminal end of the phaC encoded protein witha clamp 5′GTTC, an XbaI site TCTAGA and hybridizing region GAG TCG CTCGAA TCC TTT GTC3′. S. elodea chromosomal DNA was digested with PstI and0.5 .mu.g of DNA was ligated in a 200 .mu.l volume to allowcircularization. A KpnI digest to generate a linear DNA molecule wasused as a template in an inverse PCR reaction to generate a 1.7 kbfragment of regions flanking the 408 bp phaC fragment (SEQ ID NO: 6), asdepicted in FIG. 3.

The 1.7 kb fragment comprises the two flanking regions ligated in anorientation inverted relative to the native orientation at their PstIends. Cleavage at the PstI site indicated that the flanking regions wereof similar sizes, 850 bp and 980 bp. To reorient the fragment into itsnative orientation and, simultaneously, generate a fragment with most ofthe original 408 bp clone deleted, the 1.7 kb fragment was digested withXbaI, ligated to itself under dilute conditions to allow circularizationand then digested with PstI. The resulting fragment was cloned intoPstI-digested, CIAP-treated pUC19 and designated pEB4.

Example 3 Sequencing the phaC Clone

The 1.7 kb insert in pEB4 was sequenced and combined with the sequenceof the 408 bp fragment (SEQ ID NO: 6). The combined 1920 bp DNA sequence(SEQ ID NO: 7) is depicted in FIG. 4. Part of this sequence, from thePstI site to the TGA stop codon (bases 1-1200) encodes a protein (SEQ IDNO: 8) which is homologous to the carboxy two-thirds of other phaCgenes. Sequence alignment confirmed that the proper gene was cloned.

The phaC clone had a 232 bp deletion within the 408 bp segment and aninsertion of 6 bp, TCTAGA, corresponding to the XbaI site. The deletionand insertion caused a frameshift mutation that altered the carboxyterminus and introduced a new termination codon at base pair 1102.

Example 4 Construction of an Integration Vector and Transfer toSphingomonas by Homologous Recombination

To transfer the phaC deletion mutation into Sphingomonas elodea, a“suicide” plasmid was used, which is capable of replication in a hostsuitable for plasmid construction, for example, E. coli, but incapableof replication in Sphingomomas. Selection in Sphingomonas for theantibiotic resistance encoded by the plasmid identifies those coloniesin which the plasmid has integrated into the chromosome as a result ofhomologous recombination. Selection for the loss of antibioticresistance identifies those colonies in which the duplicated region hasrecombined out, which may result in retention of the mutation (that is,the deletion) or wild-type genes, which is depicted diagrammatically inFIG. 5. Differentiation between clones with the deletion versus cloneswith the wild-type DNA may be determined by phenotypic expression (PHBsynthesis). To measure phenotypic expression of PHB, a qualitativeturbidimetric assay was used: an aliquot of broth, about 1 ml, was addedto 9 volumes of Clorox® (Clorox Co., Oakland, Calif.) and incubated at37° C. for at least 4 hours or overnight. Appearance of a whiteprecipitate is indicative of the presence of PHB.

To facilitate detection of second crossover recombination events, apositive selection system was adapted for S. elodea. The Bacillussubtilis gene, sacB, which encodes a levansucrase, may be transferredinto gram-negative bacteria (Kamoun. S. et al., Mol. Microbiol.6:809-816 (1992); Gay, P. et al., J. Bacteriol. 164:918-921 (1985)).Growing these bacteria in sucrose promotes synthesis of levan, which istoxic to the bacteria. Consequently, if the sacB gene is present on avector, growth in sucrose may be used to identify those isolates thathave lost the vector.

The pLO2 plasmid was obtained from Steven Slater at Cereon, Monsanto.The pLO2 plasmid contains the sacB gene on a vector with kanamycinresistance, the ColEI origin of replication and the RP4 origin oftransfer as illustrated in FIG. 6 (Lenz, O. et al., J. Bacteriol.176:4385-4393 (1994)). The pLO2 plasmid may be used to transfer genesthrough the natural process of conjugation. The plasmid can replicate inE. coli, but not Sphingomonas, and contains a site for mobilization ofthe plasmid but does not contain transfer functions. That is, the pLO2plasmid is mobilizable but not self-transmissable. The genes forconjugal transfer function are supplied on a second plasmid and functionin trans. While this example uses the pLO2 plasmid, its use is notcritical. One of ordinary skill in the art would know how to design andengineer a suitable alternative plasmid and transfer it intoSphingomonas using conventional techniques, such as electroporation,transformation and the like. Similarly, use of kanamycin as a selectablemarker is not critical. One of ordinary skill in the art would know howto choose an appropriate alternative selectable marker.

The 1.7 kb PstI fragment containing the phaC deletion was ligated intoPstI-digested pLO2 and designated pLO2-phACv or pEB11 and transferredinto E. coli YMC9 (F-.DELTA.lacU169 thi endA hsdR) by transformationusing electroporation. The E. coli strain was purified and mixed withSphingomonas elodea strain S-60 wtc, along with an E. coli strain JZ279carrying plasmid pRK2013, which supplies functions for conjugal transfer(Ditta, et al., Proc. Natl. Acad. Sci. USA, 77:7347-7351 (1980)). S-60wtc is a derivative of the strain S. elodea, ATCC 31461, which wasselected as a spontaneous isolate with increased ability to take upplasmid DNA. The conjugal transfer was conducted using stationary phase(overnight) cultures, i.e. 1 ml YMC9/pLO2-phaC.DELTA., 1 mlJZ279/pRK2013 and 2-3 ml Sphingomonas elodea. Cultures were mixed andconcentrated on a filter, which was in turn placed on a TYE Petri dish(8 g/l tryptone, 5 g/l yeast extract, and 5 g/l sodium chloride) andincubated 37° C. for 7 hours. Cells were then suspended in deionizedwater and plated on selective media.

After about 7 hrs incubation, kanamycin resistant transconjugants ofS-60 wtc were selected on YM media (yeast extract 3 g/L, malt extract 3g/L, peptone 5 g/L and glucose 10 g/L) with 25 .mu.g/ml streptomycin (tocounter-select E. coli) and 7.5 .mu.g/ml kanamycin to select for theplasmid. Integration, as measured by kanamycin resistance, occurred at afrequency of 1.5.times. 10.sup.-6.

Example 5 Selection for Second Crossover Deletion Strains

Two kanamycin-resistant integrants were purified and passed three timesin non-selective YEME medium (0.25% yeast extract, 0.025% malt extract),then plated on 7.5% sucrose to select for crossouts. Sevenkanamycin-sensitive crossouts were obtained, but all were PHB-positive.A PCR test was used to verify that the vector phaCv was inserted intothe phaC region of the chromosome and to determine the location of theinsert relative to the wild-type phaC genes. Primers homologous toregions flanking the deletion and to the ends of the vector weredesigned. Recombination may occur in two orientations that result in:(1) the phaC gene with a deletion to the left and a fragment of the phaCgene to the right of the vector, which should yield a PHB-negativeclone; or (2) intact phaC gene to the left and phaCv to the right of thevector, which should yield a PHB-positive clone as depicted in FIG. 7.

Tests on six of the pLO2phaCv single crossover integrants demonstratedthat all were in the second, possibly favored, PHB-positive,orientation. There may be a strong preference for recombination on oneside of the deletion, or, alternatively, the PHB-positive strain maygrow better than the PHB-negative recombinant. Colonies in which theplasmid had integrated in the less preferred manner of the first,PHB-negative, orientation might be more likely to undergo a secondrecombination event at the preferred site resulting in a doublecrossover retaining the mutant phenotype.

The transconjugants were screened by PCR and tested for PHB expressionto identify integrants in the first, or PHB-negative, orientation. Of 24colonies tested, PCR results demonstrated that 21 were PHB-positive andthree were PHB-negative integrants. PHB tests confirmed the results. Thethree PHB-negative strains (3, 15 and 22) were selected, purified, grownfor three passages under non-selective conditions and plated on sucrose.Of five kanamycin-sensitive colonies from each parent, one wasPHB-negative. Thus, three PHB deficient, kanamycin-sensitive mutantswere isolated and designated NPG-1, NPG-2 and NPG-3.

Example 6

Characterization of Mutants for Gellan Biosynthesis 10088U NPG-1, NPG-2and NPG-3 were tested in 100 L fermentations conducted in 14 Lfermentors and compared to LPG-2, which is a PHB-deficient mutantisolated by chemical mutagenesis (U.S. Pat. No. 5,300,429). The stagesof fermentation and media used were similar to those described in U.S.Pat. No. 5,300,429, except that stage 2 medium was used for all seedstages. Three seed stages were used prior to inoculation of the finalmedium. Transfer volumes were 2.5-5%. A different organic nitrogen wasused (Quest Nzamine EKC, Chicago, Ill., at 0.41 g/L) instead of promosoyat 0.5 g/L. Corn syrup level was 3% instead of 3.75% in the seed stages.The final 10 L fermentation was similar to the seed media, but containedless phosphate (0.5 g/L K.sub.2HPO.sub.4) and the pH was controlled byaddition of KOH as required. Organic nitrogen was higher (1.1 g/L) aswas inorganic nitrogen, NaNO.sub.3 (1.5 g/L). Anti-foam H-60-K was addedto 0.6 ml/L. The corn syrup level was 3.85%. The medium in the finalstage was made up in deionized water supplemented with calcium andmagnesium.

The NPG mutants produced significantly less gellan than LPG-2, based ontotal precipitable material (“TPM”) and viscosity, as shown in Table 1.Broth viscosity was determined in a Brookfield viscometer with number 4spindle at 60 rpm. Total precipitable material was determined by heatingbroth in an autoclave for ten minutes, then precipitating with two timesvolume of isopropanol and drying. These results were reproducible.Analysis of broth samples during the fermentation indicated that a largeamount of organic acids were produced. Consequently, the low yield ofgellan for the NPG mutants correlates with a greater amount ofcarbohydrate hydrolysis to two and three carbon intermediates and carbondioxide in the absence of PHB synthesis.

TABLE 1 Summary of 10 L Fermentation Darta for NPG Strains % LPG-2 Visc.Strain (TPM) cP LPG-2 7,000 NPG-1 40 1,450 NPG-2 45 2,200 NPG-3 61 2,150

Example 7 Isolation of Mutants with Restored Gellan Productivity

The accumulation of metabolic intermediates (e.g., organic acids) due tothe blockage of PHB synthesis may have an adverse effect on gellansynthesis. It was expected that during growth in a medium that promotesgellan synthesis, a compensatory mutation could occur that allows gellansynthesis to proceed at normal levels. Aliquots of fermentation brothfrom the 10 L fermentations (Example 6), were plated to determine cellcounts (FIG. 8). It was observed that towards the end of thefermentation (i.e., at 44 and 69 hours) between about 0.5% and 2% of thecolonies were larger and more mucoid than the NPG strains. Thesecolonies were purified and tested for PHB and gellan production in shakeflask fermentations. The new isolates were PHB-deficient and had ahigher yield of gellan than the original NPG mutants. The bestcompensatory mutants had total precipitable material comparable to LPG-2and .gtoreq.80% of the wild type (an approximate 10-15% decrease in TPMis expected due to the loss of weight of PHB.). These strains weredesignated PDG mutants: PDG-1 is derived from NPG-1, and PDG-3 isderived from NPG-3. Each of the strains was evaluated in shake flaskfermentations for PHB and gellan production.

TABLE 2 Shake Flask Fermentation for PHB-Deficient Strains TPM g/100Strain ml % S60 % LPG2 PHB S60-wtc 1.52 + LPG-2 1.42 93 − NPG-1 0.72 4750 − PDG-1 1.44 94 102  − NPG-3 0.51 34 36 − PDG-3 1.37 90 96 −

In addition, broth viscosities of a second batch of S60-wtc, LPG-2,PDG-1 and PDG-3 were determined using a Brookfield viscometer withnumber 4 spindle at 60 rpm. The broth viscosities are shown in Table 3below.

TABLE 3 Experiment 2 Broth Viscosities f PHB-Deficient Strains Visc.Strain cP^(a) × 10³ S60-wtc 84 LPG-2 92 PDG-1 83 PDG-3 63

The media used for shake flasks was similar to that described in U.S.Pat. No. 5,300,429. The first seed contained YM medium. Second and finalstages were 100 ml per 500 ml shake flasks, containing medium asdescribed previously, but with higher phosphate for buffering(K.sub.2HPO.sub.4, 2.8 g/L; KH.sub.2PO.sub.4, 1.2 g/L) and organicnitrogen at 1.0 g/L.

Without being bound by theory, the new mutations may be spontaneousmutants which limit the breakdown of glucose to organic acids. Analysisof in-cycle samples from fermentors indicated that production of organicacids with PDG-1 and PDG-3 was about the same as that of the controlstrains, S-60 wtc and LPG-2.

Strain PDG-1 consistently produced good yield of high viscosity gellanwith a TPM>14 g/L.

Colony morphology on plates of these cultures was evaluated to checkstability of the strains and particularly, to compare the spontaneousPDG-1 mutants to the original NPG strains which had low gellan yields.After growth on YM agar at about 37° C. for about 60 hours, PDG-1 showeddistinctly different morphology than its parent NPG-1. Colonies withNPG-1 type morphology were not observed in broth from PDG-1fermentations, which indicates the stability of the strain.

Example 8 Presence of Homologous phaC Genes in Sphingomonas StrainsOther than S. Elodea

Genes homologous to phaC were identified in strains of Sphingomonasother than Sphingomonas elodea thereby demonstrating the feasibility ofgenerating PHB-deficient mutants in strains of Sphingomonas other thanSphingomonas elodea.

Southern DNA hybridization was conducted with four Sphingomonas strains:ATCC 53159, which produces diutan (S-657); ATCC 31555, which produceswelan (S-130); ATCC 31961, which produces rhamsan (S-194); and ATCC31461, which produces gellan (S-60) as control. Genomic DNA was isolatedfrom each strain and digested with the enzyme EcoRI. Samples of digestedgenomic DNA (1 .mu.g) were separated on a 1% agarose gel and transferredto nylon membranes via capillary action using a Schleicher and SchuellTurboblotter® (Keene, N.H.) under neutral conditions.

Using degenerate primers PHADG5 and PHADG7 (see example 1), adigoxigenin-labeled probe was prepared by PCR-amplification of aninternal region of the Sphingomonas S-657 phaC gene withdigoxigenin-11-dUTP according to the protocol of the manufacturer, RocheMolecular Biochemicals, Switzerland. Hybridization was conducted underneutral conditions using DigEasyHyb® from Roche Diagnostics (Mannheim,Germany) according to the protocol of the manufacturer. The filters werehybridized at 44° C., which is 10° C. lower than the calculatedT.sub.opt. These conditions are expected to result in hybridization ofDNA molecules that are more than 90% identical (Birren, B., et al., Eds.Genomic Analysis, A Laboratory Manual, (1997). As used herein, the termT.sub.opt is defined as T.sub.m⁻²⁰, where T.sub.m is defined by theformula 50+0.41(% GC)−600/probe length, where the % GC is 65% and theprobe length is 400 nucleotides. The filters were washed in 2.times.SSC, 0.1% SDS two times for 15 minutes at 44° C. and developed using ananti-digoxigenin-alkaline phosphatase conjugate and a digoxigenindetection kit according to the manufacturer's protocol (Roche MolecularBiochemicals).

The results of the hybridization are shown in FIG. 9. An EcoRI-digestedband of the expected size (2.6 kB) was detected in the Sphingomonasstrains ATCC 31461. Sphingomonas strains ATCC 53159 and ATCC 31961produced a band of exactly the same size. Sphingomonas ATCC 31555contained a 2.4 kB fragment that hybridized to the phaC probe. Thus, theSouthern DNA hybridization confirmed that these three strains contain aphaC-like gene and that PHB-deficient strains could be generatedaccording to the methods described herein.

Example 9 Construction of Mutant Strains of ATCC 53159 Having phaCDeletions

Using recombinant DNA techniques, mutant strains of Sphingomonas ATCC53159 were constructed in which the phaC gene was completely deleted.The construction of the mutant strain was performed as follows: DNAregions flanking the phaC gene were amplified by PCR and cloned into asuicide vector, the suicide vector containing the flanking PCR productswas transferred by conjugation into ATCC 53159 cells, then integrationof the entire plasmid at a homologous locus directly upstream ordownstream of phaC in the Sphingomonas ATCC 53159 chromosome wasachieved by selection for kanamycin resistance (as encoded by thevector). Excision of the phaC locus plus the vector DNA from thechromosome was a result of a subsequent second cross-over event whichwas selected for by sucrose-sensitivity encoded on the vector.

To isolate clones containing the phaC gene and flanking regions, genomicDNA libraries were prepared and screened by PCR, using PHADG5 and PHADG7primers (see Example 1 above). Two genomic libraries were made, one withNotI restriction enzyme fragments in vector pZERO-2 (Invitrogen,Carlsbad, Calif.), the second with Sau3A partial digest fragments inpLAFR3 (Staskawicz et al. J. Bacteriol. 169:5789-94 (1987)). Onepositive clone was isolated from each library. BamHI-NotI fragments fromthese plasmids were subcloned and appropriate fragments sequenced todetermine the DNA sequence of the phaC gene and the 5′ and 3′ flankingregions. Plasmids p21-7 and pJCS104-2 contain respectively, the 5′ and3′ ends of the phaC gene and flanking regions.

The DNA sequences of the phaC gene and flanking regions are shown inFIG. 10. A genetic map is shown in FIG. 11. Open reading frames weredetermined by the presence of start and stop codons and BLAST analysiscombined with the predicted coding regions using Borodovsky analysis(Lasergene GeneQuest module) and the P_aeruginosa.sub.—3.mat matrix fromGeneMark. The sequence is linked from the insert sequences in clonesp21-7 and pJCS104-2. The junction between the two sequences is at theNotI site.

FIG. 12 depicts how PCR was used to make a product pJCS105 112-1 thatcontains only the regions flanking phaC, deleting the entire phaC gene(1737 bp) from the first nucleotide of the start codon to the lastnucleotide of the stop codon. Two outer primers (primer 1Xba and primer4Xba) were combined with a primer (overlap1) that spans the desiredjunction between the upstream and downstream regions of phaC. Primersequences are shown in Table 4.

TABLE 4 Primer Sequences for ATCC 53159 phaC deletionNucleotides (5′_3′) Primer-binding Restriction Site Primer(Restriction enzyme site underlined) Sites Added primerATTCTAGAGATGATGAAGCCGAAGGTGTGGAT 537 bp upstream XbaI 1Xba(SEQ ID NO: 14) of phaC primer ATTCTAGATGGTGCGCTCGTTGAGG 512 bp XbaI4Xba1 (SEQ ID NO: 15) downstream of phaC overlapGAAATTCTGCCTCTTTGTCGGTCCTCTCCTTCGC spans the phaC none 1 (SEQ ID NO: 16)gene open reading frame

Plasmids pJCS104-2 and p21-7 (200 ng each) were mixed with primers 1Xbaand 4Xba (50 pmol each), the overlap1 primer (2 pmol), dNTPs and Taqpolymerase from Advantage High Fidelity 2 PCR kit from ClontechLaboratories, Inc. (Palo Alto, Calif.) as per manufacturer's protocol.Amplification was then conducted for 1 min 95° C., 5 cycles of 30 sec at95C, 30 sec 44° C., 2 min 68° C., then 20 cycles of 30 sec 95° C., 30sec 53-68° C., 2 min 68° C., followed by single cycle of 3 min 68° C.,in a Matrix thermocycler. The amplified DNA fragment was purified from agel and further amplified with the same primers to produce more product.The amplification conditions were 1 min 95° C., 25 cycles of 30 sec at95° C., 30 sec 64° C. 2 min 68C, then a single cycle at 68° C. A 1.1 kBband was then isolated from the gel using SNAP gel Purification Kit®(Invitrogen) and cloned into vector pCRII-TOPO (Invitrogen) by usingtopoisomerase, a vector with 3′ T overhangs and chemically competentTOP10 cells, according to Invitrogen protocol, to form pJC105-112-1 asshown in FIG. 12.

The 1.1 kB XbaI fragment containing the phaC deletion construct frompJCS105 112-1 was gel purified and cloned into XbaI-digested pLO2. Twoorientation of the insert were recovered and designated pJCS106-5 andpJC106-16.

Marker exchange was used to make a PHB-deficient strain of ATCC 53159Plasmids pJCS106-5 and pJC106-16 were introduced to ATCC 53159 bytransconjugation, as per Example 4 above. Selection for the first andsecond crossover deletion strains proceeded as by examples 4 and 5above, i.e., selecting first for integration as shown by kanamycinresistance and then plating on sucrose to select for kanamycin sensitivecrosscuts. The deletion crossouts (versus wild-type) were detected bydiagnostic PCR, and designated NPD-3 (derived from pJCS106-5) and NPD-6(derived from pJCS106-16.

The resulting NPD-3 and NPD-6 strains have a precise chromosomaldeletion of phaC with no foreign DNA remaining. The gum yields of thesedeletion strains were greatly reduced however, but suppressors thatrestored gum production were subsequently isolated upon growing infermentation.

NPD-3 and NPD-6 were grown under conditions to promote S-657 synthesis,and suppressor strains having large, mucoid colonies were selected, aswas done for gellan synthesis in Example 7 above. These large, mucoidcolonies were designated PDD-3 and PDD-6, as per the colonies from whichthey were derived, and were analyzed for PHB and S-657 production. ThePDD-3 and PDD-6 strains are on deposit with the American Type CultureCollection and designated as ATCC No. PTA-4865 and ATCC No. PTA-4866,respectively both deposited on Dec. 20, 2002. Table 5 below, indicatesthat PDD-6 provided greater gum production than its predecessor NPD-6strain while also qualitatively producing no PHB.

TABLE 5 Fermentation Results for S-657 PHB-Deficient Strains TPM Straing/L S657 20.7 + NPD-6  5.6 27% − PDD-6 18.7 90% −

Example 10 Effect of Potassium Hydroxide Concentration on Transmittanceand Gel Strength

The effect of the concentration of the caustic agent potassium hydroxide(“KOH”) was assessed in the clarification process comprising the stepsoutlined above as the first protocol. A gellan fermentation brothcomprising a PHB-deficient mutant was pretreated and mixed with varyingconcentrations of KOH for 15 min, followed by 1000 ppm Calgon aschelating agent for 1 hr, followed sequentially by 22 ppm lysozyme and220 ppm protease for 2 hrs each at 55° C. The KOH concentration testedvaried between about 0.0 g/L and about 0.45 g/L. As depicted in FIG. 13,transmittance increased nearly 20% (31% relative increase) as theconcentration of KOH increased to 0.45 g/L.

The TA-TX2 Texture Analyzer® (Texture Technologies Corp., Scarsdale,N.Y.) measures gel strength data as a product of two indicators, theamount of puncture force and distance required to fracture a preparedgel surface with a pressure-sensing plunger. Puncture force isdetermined when a load cell detects a break in the gel surface, andpuncture force is determined as a percent change in height. As depictedin FIG. 14, gel strength with respect to puncture force decreased 280 g,or 32%, over the same range of KOH tested, which may be attributed tothe partial deacylation of the gellan. However, gel strength withrespect to percent distance did not seem to be significantly affected,reflecting only a 1.5% change.

A small 2×2 factorial study was conducted according to the firstprotocol clarification process. A gellan fermentation broth comprising aPHB-deficient mutant was pretreated and mixed with varyingconcentrations of KOH for 15 min, followed by 2000 ppm Calgon aschelating agent for 1 hr, followed sequentially by 22 ppm lysozyme and220 ppm protease for 2 hrs each at 55° C. In this study, percenttransmittance, puncture force and percent distance were studied becausethey are believed to be interrelated to kinetics. The KOH concentrationvaried between about 0.225 g/L and about 0.45 g/L, and the temperaturevaried between about 55° C. and about 60° C. to produce the resultsshown in the following tables, which demonstrate the percenttransmittance, puncture force and percent distance results evaluatingthe effect of KOH concentration and temperature on gellan clarification.

TABLE 3 [KOH] [KOH] 0.225 0.45 Temp. g/L g/L Percent transmission 55° C.78.05 77.9 60° C. 80.9 84.2 Puncture Force 55° C. 753 562 60° C. 600 428Distance 55° C. 89.2 87.2 60° C. 90.7 88.2

As demonstrated in the tables, the transmittance did not change muchupon increasing either the KOH concentration or temperature separately.However, transmittance increased by about 6% when both KOH concentrationand temperature were increased, which indicates that both parameters arecritical and additive for achieving increased transmittance. Similarly,gel strength exhibited an additive effect. Puncture force decreased byabout 130 g to about 190 g upon increasing either the temperature or KOHconcentration individually; however, upon increasing both temperatureand KOH concentration, the puncture force was reduced by about 326 g,thus suggesting that gel strength is susceptible to changes in bothtemperature and KOH concentration.

Example 11 Effect of Sodium Hexametaphosphate on Gellan Properties

The effect of sodium hexametaphosphate (“SHMP”), which is also known asCalgon, on transmittance, puncture force and percent distance wasevaluated according to the clarification process described in Example 10above. A gellan fermentation broth comprising a PHB-deficient mutant waspretreated and mixed with 0.45 g/L KOH for 15 min, followed by varyingconcentrations of Calgon for 1 hr, followed sequentially by 22 ppmlysozyme and 220 ppm protease for 2 hrs each at 55° C. SHMPconcentration varied between about 1000 ppm and about 3000 ppm, and asdemonstrated in FIG. 15, a linear correlation exists between SHMPconcentration and transmittance over this range. An increase of about1000 ppm SHMP results in an about 5% increase in transmittance. As shownin FIG. 16, SHMP does not appear to affect gel strength because bothpuncture force and percent distance are relatively unaffected by theincrease in SHMP concentration over the range tested.

Example 12 Alternative Clarification Sequences with SHMP

Two variations of the clarification method were conducted on nativegellan broth at the 2 L scale. According to information supplied by themanufacturer Genencor International, Inc. (Rochester, N.Y.), Multifect®lysozyme is stable at acidic to neutral pH levels and can be inactivatedat alkaline pH within a short period of time. After addition of 0.45 g/LKOH according to the clarification process, the pH generally exceeds pH8, which is sub-optimal for lysozyme, while protease purportedly workswell under these conditions. Thus, the clarification process wasmodified as per the second protocol, to add KOH after the treatment withlysozyme enzyme (sequence summary: lysozyme, then KOH, then protease).Whether KOH was added before or after lysozyme enzyme treatment, a 5.5%relative standard deviation (“RSD”) was observed, as shown in thefollowing table.

TABLE 5 Trans Force Dist. Protocol; [SHMP] (%) (g) (%) N= Mean Firstprotocol; 82.38 436.85 87.84 4 RSD 2000 ppm SHMP 5.50  0.28  0.02 MeanSecond protocol; 81.34 **** **** 6 RSD 2000 ppm SHMP 5.40 Mean Secondprotocol; 83.83 **** **** 3 RSD 1500 ppm SHMP 7.10 ****refers tounmeasured data RSD refers to relative standard deviation as apercentage of the mean.

Example 13 Confection Formulation

This example demonstrates a formulation that may be used to produce anelastic and resilient chewy confection that exhibits excellent clarityand stability.

Ingredients Percent Part A Glucose syrup 45.00 Water 21.67 Part BSucrose 30.00 Clarified high-acyl gellan 1.33 Kelcogel F ® gellan 0.67(CP Kelco U.S., Inc., San Diego, CA) Part C Citric acid solution, 54%0.67 Sodium citrate solution, 33% 0.67

The components comprising Part A were combined in a heating vessel andheated to 40° C.

The components of Part B were blended dry and added to the heatingvessel mixed rapidly, and brought to boil. The mixture was reduced to72% solids. The components of Part C were combined and added to flavorand color, and mixed until homogeneous.

The material was placed into a depositer and casted into prepared starchmolds. The filled starch molds were then stored at 30° C. and 35%relative humidity for 3 to 4 days until the solids level reached betweenabout 82% and 85%. The material was de-molded, waxed and stored insealed bags.

Additional water may be added to the Part A ingredients to facilitatecomplete hydration of the hydrocolloids.

Example 14 Dessert Gel Formulation

This formulation was used to produce an elastic and resilient dessertgel with excellent clarity and stability.

Ingredients Percent Part A Sucrose 13.20 Adipic acid 0.40 Clarified,high-acyl gellan 0.16 Sodium citrate 0.13 Disodium phosphate 0.13Fumaric Acid 0.11 Kelcogel F ® gellan 0.04 (CP Kelco U.S., Inc., SanDiego, CA) Part B Water 85.83

The ingredients of Part A, in addition to dry flavor and color, wereblended dry and dispersed into Part B and mixed; heated to 90° C. Themixture was then poured into suitable containers and allowed to set atroom temperature.

Example 15 Jelly Formulation

This formulation provided a jelly with excellent clarity, storagestability, flavor release and spread-ability.

Ingredients Percent Part A Concord grape juice 45.69 High fructose cornsyrup 30.46 Water 22.85 Part B Clarified, high-acyl gellan 0.18 Sodiumcitrate 0.10 SHMP 0.10 Potassium sorbate 0.09 Part C Citric Acid, 50%solution 0.58

The ingredients of Part A were combined. The ingredients of Part B wereblended dry and dispersed with the ingredients of Part A while mixing.The resulting mixture was brought to boil while mixing and held at aboil for about 1 to about 3 minutes, at which point, the ingredients ofPart C were stirred into the mixture. The mixture was then depositedinto sterilized jars and sealed.

While the present invention is described above with respect to what iscurrently considered to be its preferred embodiments, it is to beunderstood that the invention is not limited to that described above. Tothe contrary, the invention is intended to cover various modificationsand equivalent arrangements included within the spirit and scope of theappended claims.

The invention claimed is:
 1. A process of preparing a clarified diutan product of native diutan produced by a Sphingomonas mutant strain genetically modified from a wild-type Sphingomonas strain ATCC 53159, wherein a phaC gene in said Sphingomonas mutant strain has been selectively mutated such that the mutant strain produces native diutan without producing polyhydroxybutyrate (PHB) due to lack of expression of phaC protein in said mutant strain, comprising the steps of: a) heating an aqueous diutan solution containing native diutan of the mutant strain to a clarification temperature of 30-70° C., b) treating the heated aqueous diutan solution from step a) with at least one chelating agent, c) treating the diutan solution from step b) with a lysozyme enzyme, a protease, or both, and d) recovering the clarified diutan product by precipitation with an alcohol.
 2. The clarification process of claim 1, wherein the at least one chelating agent is selected from the group consisting of ethylenediamine tetraacetic acid, phosphoric acid, metaphosphoric acid, carbonic acid, citric acid, tartaric acid, gluconic acid, glutamic acid, pyrophosphoric acid, polyphosphoric acid, metaphosphoric acids, saccharic acid, ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), ethylenediamine, 2,3-diaminobutane, 1,2-diaminocyclohexane, triaminotriethylamine, and a salt thereof.
 3. The clarification process of claim 1, wherein the at least one chelating agent is selected from the group consisting of disodium ethylenediamine tetraacetate, dipotassium ethylenediamine tetraacetate, tetrasodium ethylenediamine tetraacetate, tetrapotassium ethylenediamine tetraacetate, trisodium citrate, tripotassium citrate, sodium hexametaphosphate, potassium hexametaphosphate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, monosodium phosphate, monopotassium phosphate, disodium phosphate, dipotassium phosphate, trisodium phosphate, tripotassium phosphate, sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate, a cationic ion exchange resin, ethylenediamine dihydrochloride, ethylenediamine diacetate, ethylenediamine lithium salt, ethylenediamine dihydroiodide, and mixtures thereof.
 4. The clarification process of claim 1, further comprising a step of treating the diutan solution from step c) with at least one caustic agent, an oxidizing agent, or both.
 5. The clarification process of claim 4, wherein the caustic agent is selected from the group consisting of potassium hydroxide, sodium hydroxide and trisodium phosphate.
 6. The clarification process of claim 4, wherein the oxidizing agent is selected from the group consisting of sodium hypochlorite or other hypochlorite salts, chloride dioxide, hydrogen peroxide, peracetic acid and ozone.
 7. The clarification process of claim 1, further comprising a step of treating the diutan solution from step a) with a surfactant during or after the chelating agent treatment in step b).
 8. The clarification process of claim 7, wherein the surfactant is selected from the group consisting of SDS, polyoxyethylenesorbitan monooleate, lecithin, a monoglyceride, a tartaric ester of a monoglyceride, a phosphated monoglyceride, a lactylated monoglyceride, an acetylated monoglyceride, a succinylated monoglyceride, an ethoxylated monoglyceride, a sorbitan ester, a polysorbate, a polyglycerol ester, a sucrose ester, a sodium stearoyl lactylate, and a propylene glycol ester.
 9. The clarification process of claim 1, wherein the treatment with the lysozyme enzyme is conducted at a pH of 3 to 7.5.
 10. The clarification process of claim 1, wherein the treatment with the protease enzyme is conducted at a pH of 6.5 to
 9. 